Cytokine receptor chain

ABSTRACT

The present invention is directed to methods for treating asthma.

This application is a continuation-in-part of application Ser. No.08/841,751, filed Apr. 30, 1997, which was a divisional application ofapplication Ser. No. 08/609,572, filed Mar. 1, 1996.

FIELD OF THE INVENTION

The present invention relates to mammalian cytokine receptor proteinswith affinity for IL-13 (including without limitation human and murinereceptor proteins), fragments thereof and recombinant polynucleotidesand cells useful for expressing such proteins.

BACKGROUND OF THE INVENTION

A variety of regulatory molecules, known as cytokines, have beenidentified including interleukin-13 (IL-13). Various protein forms ofIL-13 and DNA encoding various forms of IL-13 activity are described inMcKenzie et al., Proc. Natl. Acad. Sci. USA 90:3735 (1993); Minty etal., Nature 362:248 (1993); and Aversa et al., WO94/04680. Thus, theterm “IL-13” includes proteins having the sequence and/or biologicalactivity described in these documents, whether produced by recombinantgenetic engineering techniques; purified from cell sources producing thefactor naturally or upon induction with other factors; or synthesized bychemical techniques; or a combination of the foregoing.

IL-13 is a cytokine that has been implicated in production of severalbiological activities including: induction of IgG4 and IgE switching,including in human immature B cells (Punnonen et al., J. Immunol.152:1094 (1994)); induction of germ line IgE heavy chain (ε)transcription and CD23 expression in normal human B cells (Punnonen etal., Proc. Natl. Acad. Sci. USA 90:3730 (1993)); and induction of B cellproliferation in the presence of CD40L or anti-CD40 mAb (Cocks et al.,Int. Immunol. 5:657 (1993)). Although many activities of IL-13 aresimilar to those of IL-4, in contrast to IL-4, IL-13 does not havegrowth promoting effects on activated T cells or T cell clones (Zurawskiet al., EMBO J. 12:2663 (1993)).

Like most cytokines, IL-13 exhibits certain biological activities byinteracting with an IL-13 receptor (“IL-13R”) on the surface of targetcells. IL-13R and the IL-4 receptor (“IL-4R”) sharing a commoncomponent, which is required for receptor activation; however, IL-13does not bind to cells transfected with the 130 kD IL-4R (Zurawski etal., supra). Thus, the IL-13R must contain at least one other ligandbinding chain. Cytokine receptors are commonly composed or two or threechains. The cloning of one ligand binding chain for IL-13 has beenrecently reported (Hilton et al., Proc. Natl. Acad. Sci. 93:497-501).

It would be desirable to identify and clone the sequence for any otherIL-13 binding chain of IL-13R so that IL-13R proteins can be producedfor various reasons, including production of therapeutics and screeningfor inhibitors of IL-13 binding to the receptor and receptor signaling.

SUMMARY OF THE INVENTION

In accordance with the present invention, polynucleotides encoding theIL-13 binding chains of the interleukin-13 receptor are disclosed,including without limitation those from the murine and human receptors.In certain embodiments, the invention provides an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of:

-   -   (a) the nucleotide sequence of SEQ ID NO: 1 from nucleotide 256        to nucleotide 1404;    -   (b) the nucleotide sequence of SEQ ID NO:3 from nucleotide 103        to nucleotide 1242;    -   (c) a nucleotide sequence varying from the sequence of the        nucleotide sequence specified in (a) or (b) as a result of        degeneracy of the genetic code;    -   (d) a nucleotide sequence capable of hybridizing under stringent        conditions to the nucleotide specified in (a) or (b);    -   (e) a nucleotide sequence encoding a species homologue of the        sequence specified in (a) or (b); and    -   (f) an allelic variant of the nucleotide sequence specified        in (a) or (b). Preferably, the nucleotide sequence encodes a        protein having a biological activity of the human IL-13        receptor. The nucleotide sequence may be operably linked to an        expression control sequence. In preferred embodiments, the        polynucleotide comprises the nucleotide sequence of SEQ ID NO:1        from nucleotide 256 to nucleotide 1404; the nucleotide sequence        of SEQ ID NO:1 from nucleotide 319 to nucleotide 1257; the        nucleotide sequence of SEQ ID NO:1 from nucleotide 1324 to        nucleotide 1404; the nucleotide sequence of SEQ ID NO:3 from        nucleotide 103 to nucleotide 1242; the nucleotide sequence of        SEQ ID NO:3 from nucleotide 178 to nucleotide 1125; or the        nucleotide sequence of SEQ ID NO:3 from nucleotide 1189 to        nucleotide 1242.

The invention also provides isolated polynucleotides comprising anucleotide sequence encoding a peptide or protein comprising an aminoacid sequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:2;    -   (b) the amino acid sequence of SEQ ID NO:2 from amino acids 22        to 334;    -   (c) the amino acid sequence of SEQ ID NO:2 from amino acids 357        to 383;    -   (d) the amino acid sequence of SEQ ID NO:4;    -   (e) the amino acid sequence of SEQ ID NO:4 from amino acids 26        to 341;    -   (f) the amino acid sequence of SEQ ID NO:4 from amino acids 363        to 380; and    -   (g) fragments of (a)-(f) having a biological activity of the        IL-13 receptor binding chain. Other preferred embodiments encode        the amino acid sequence of SEQ ID NO:2 from amino acids 1 to 331        and the amino acid sequence of SEQ ID NO:2 from amino acids 26        to 331.

Host cells, preferably mammalian cells, transformed with thepolynucleotides are also provided.

In other embodiments, the invention provides a process for producing aIL-13bc protein. The process comprises:

-   -   (a) growing a culture of the host cell of the present invention        in a suitable culture medium; and    -   (b) purifying the human IL-13bc protein from the culture.        Proteins produced according to these methods are also provided.

The present invention also provides for an isolated IL-13bc proteincomprising an amino acid sequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:2;    -   (b) the amino acid sequence of SEQ ID NO:2 from amino acids 22        to 334;    -   (c) the amino acid sequence of SEQ ID NO:2 from amino acids 357        to 383;    -   (d) the amino acid sequence of SEQ ID NO:4;    -   (e) the amino acid sequence of SEQ ID NO:4 from amino acids 26        to 341;    -   (f) the amino acid sequence of SEQ ID NO:4 from amino acids 363        to 380; and    -   (g) fragments of (a)-(f) having a biological activity of the        IL-13 receptor binding chain        Preferably the protein comprises the amino acid sequence of SEQ        ID NO:2; the sequence from amino acid 22 to 334 of SEQ ID NO:2;        the sequence of SEQ ID NO:4; or the sequence from amino acid 26        to 341 of SEQ ID NO:4. In other preferred embodiments, the        specified amino acid sequence is part of a fusion protein (with        an additional amino acid sequence not derived from IL-13bc).        Preferred fusion proteins comprise an antibody fragment, such as        an Fc fragment. Particularly preferred embodiments comprise the        amino acid sequence of SEQ ID NO:2 from amino acids 1 to 331 and        the amino acid sequence of SEQ ID NO:2 from amino acids 26 to        331.

Pharmaceutical compositions comprising a protein of the presentinvention and a pharmaceutically acceptable carrier are also provided.

The present invention further provides for compositions comprising anantibody which specifically reacts with a protein of the presentinvention.

Methods of identifying an inhibitor of IL-13 binding to the IL-13bc orIL-13 receptor are also provided. These methods comprise:

-   -   (a) combining an IL-13bc protein or a fragment thereof with        IL-13 or a fragment thereof, said combination forming a first        binding mixture;    -   (b) measuring the amount of binding between the protein and the        IL-13 or fragment in the first binding mixture;    -   (c) combining a compound with the protein and the IL-13 or        fragment to form a second binding mixture;    -   (d) measuring the amount of binding in the second binding        mixture; and    -   (e) comparing the amount of binding in the first binding mixture        with the amount of binding in the second binding mixture;        wherein the compound is capable of inhibiting IL-13 binding to        the IL-13bc protein or IL-13 receptor when a decrease in the        amount of binding of the second binding mixture occurs.        Inhibitors of IL-13R identified by these methods and        pharmaceutical compositions containing them are also provided.

Methods of inhibiting binding of IL-13 to the IL-13bc proteins or IL-13receptor in a mammalian subject are also disclosed which compriseadministering a therapeutically effective amount of a compositioncontaining an IL-13bc protein, an IL-13bc or IL-13R inhibitor or anantibody to an IL-13bc protein.

Methods are also provided for potentiating IL-13 activity, whichcomprise combining a protein having IL-13 activity with a protein ofclaim 11 and contacting such combination with a cell expressing at leastone chain of IL-13R other than IL-13bc. Preferably, the contacting stepis performed by administering a therapeutically effective amount of suchcombination to a mammalian subject.

Further methods are provided for treating an IL-13-related condition ina mammalian subject, said method comprising administering atherapeutically effective amount of a composition comprising an IL-13antagonist and a pharmaceutically acceptable carrier. Other methodsprovide for a method of inhibiting the interaction of IL-13 with anIL-13bc protein in a mammalian subject comprising administering atherapeutically effective amount of a composition comprising an IL-13antagonist and a pharmaceutically acceptable carrier. Preferably, theantagonist is selected from the group consisting of an IL-13bc protein,a soluble form of IL-13Rα1, an antibody to IL-13 or an IL-13-bindingfragment thereof, an antibody to IL-13bc or an IL-13bc-binding fragmentthereof, an antibody to IL-13Rα1 or an IL-13Rα1-binding fragmentthereof, IL-13R-binding mutants of IL-4, a small molecule capable ofinhibiting the interaction of IL-13 with IL-13bc and a small moleculecapable of inhibiting the interaction of IL-13 with IL-13Rα1.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1: The figure presents photographs of IL-13, IL-4, IL-11 and mocktransfected COS cells after exposure to IL-13bc-Fc as described inExample 4 below.

FIG. 2: Reversal of allergen-induced airway hyper responsiveness by invivo blockade of interleukin-13. 10 days after initial intratrachealchallenge, OVA- and PBS-immunized mice were again challengedintratracheally with either OVA or PBS. Mice were given sIL-13bc-Fc (400ug) or an equivalent amount of control hu-IgG by intraperitonealinjection on Day −1, O, +1 and +3 of the secondary antigen challenge.The allergic phenotype was assessed 4 days after the PBS or OVAchallenge. (A) Airway hyper responsiveness (AHR) to acetylcholinechallenge, defined by the time-integrated rise in peak airway pressure(airway-pressure-time index [APTT] in cmH₂O×sec). (B) Inflammatory cellcomposition of bronchoalveolar lavage fluids. Cell differentialpercentages were determined by light microscopic evaluation of cytospinpreparations. Data are expressed as absolute numbers of cells. (C)OVA-specific serum IgE concentrations. Results are means +/−SEM of 8-10animals per group. *P<0.05 compared with respective PBS control groups;**P<0.05 compared to OVA/Hu-Ig group (one-way ANOVA followed by Fisher'sleast significant difference test for multiple comparisons).

FIG. 3: Effects of IL-13 blockade on allergen-driven increases inmucus-containing cells in the airway epithelium. Lung sections (N=4 perexperimental group, four sections per animal) were fixed in formalin,cut into 10 um sections and stained with hematoxylin and eosin, andperiodic acid Schiff. Representative sections are shown. Bars=100 um.PBS/Hu-Ig: PBS-immunized and challenged controls, demonstrating fewmucus-containing cells. OVA/Hu-Ig: allergen-induced increases ininterstitial inflammatory cells and increases in the number of gobletcells containing mucus. OVA/sIL-13bc-Fc: dramatic inhibitory effect ofIL-13 blockade on allergen-induced goblet cell mucus production.

FIG. 4: IL-13 induction of airway hyperreactivity. Naive mice were givenrecombinant IL-13 (5 ug/mouse, 50 ul volume) or PBS daily byintratracheal instillation. 24 hrs after the last treatment, (A) Airwayhyper responsiveness, (B) BAL eosinophil levels, (C) Serum total IgElevels, and (D) Mucus score were determined. Results are means +/−SEM(vertical bars) of 7-10 animals per group. *P<0.05 compared to PBS group(Student's t test).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The inventors of the present application have for the first timeidentified and provided polynucleotides encoding the IL-13 binding chainof IL-13R (hereinafter “IL-13bc”), including without limitationpolynucleotides encoding murine and human IL-13bc.

SEQ ID NO:1 provides the nucleotide sequence of a cDNA encoding themurine IL-13bc. SEQ ID NO:2 provides predicted the amino acid sequenceof the receptor chain, including a putative signal sequence from aminoacids 1-21. The mature murine IL-13bc is believed to have the sequenceof amino acids 22-383 of SEQ ID NO:2. The mature murine receptor chainhas at least three distinct domains: an extracellular domain (comprisingapproximately amino acids 22-334 of SEQ ID NO:2), a transmembrane domain(comprising approximately amino acids 335-356 of SEQ ID NO:2) and anintracellular domain (comprising approximately amino acids 357-383 ofSEQ ID NO:2).

SEQ ID NO:3 provides the nucleotide sequence of a cDNA encoding thehuman IL-13bc. SEQ ID NO:4 provides predicted the amino acid sequence ofthe receptor chain, including a putative signal sequence from aminoacids 1-25. The mature human IL-13bc is believed to have the sequence ofamino acids 26-380 of SEQ ID NO:4. The mature human receptor chain hasat least three distinct domains: an extracellular domain (comprisingapproximately amino acids 26-341 of SEQ ID NO:4), a transmembrane domain(comprising approximately amino acids 342-362 of SEQ ID NO:4) and anintracellular domain (comprising approximately amino acids 363-380 ofSEQ ID NO:4).

The first 81 amino acids of the human IL-13bc sequence are identical tothe translated sequence of an expressed sequence tag (EST) identified as“yg99f10.r1 Homo sapiens cDNA clone 41648 5′” and assigned databaseaccession number R52795.gb_est2. There are no homologies or sequencemotifs in this EST sequence which would lead those skilled in the art toidentify the encoded protein as a cytokine receptor. A cDNA clonecorresponding to this database entry is publicly-available from theI.M.A.G.E. Consortium. Subsequent to the priority date of the presentapplication, such clone was ordered by applicants and sequenced. Thesequence of such clone was determined to be the sequence previouslyreported by applicants as SEQ ID NO:3 herein.

Soluble forms of IL-13bc protein can also be produced. Such solubleforms include without limitation proteins comprising amino acids 1-334or 22-334 of SEQ ID NO:2 or amino acids 1-341 or 26-341 of SEQ ID NO:4.The soluble forms of the IL-13bc are further characterized by beingsoluble in aqueous solution, preferably at room temperature. IL-13bcproteins comprising only the intracellular domain or a portion thereofmay also be produced. Any forms of IL-13bc of less than full length areencompassed within the present invention and are referred to hereincollectively with full length and mature forms as “IL-13bc” or “IL-13bcproteins.” IL-13bc proteins of less than fill length may be produced byexpressing a corresponding fragment of the polynucleotide encoding thefull-length IL-13bc protein (SEQ ID NO:1 or SEQ ID NO:3). Thesecorresponding polynucleotide fragments are also part of the presentinvention. Modified polynucleotides as described above may be made bystandard molecular biology techniques, including construction ofappropriate desired deletion mutants, site-directed mutagenesis methodsor by the polymerase chain reaction using appropriate oligonucleotideprimers.

For the purposes of the present invention, a protein has “a biologicalactivity of the IL-13 receptor binding chain” if it possess one or moreof the following characteristics: (1) the ability to bind IL-13 or afragment thereof (preferably a biologically active fragment thereof);and/or (2) the ability to interact with the second non-IL-13-bindingchain of IL-13R to produce a signal characteristic of the binding ofIL-13 to IL-13R. Preferably, the biological activity possessed by theprotein is the ability to bind IL-13 or a fragment hereof, morepreferably with a K_(D) of about 0.1 to about 100 nM. Methods fordetermining whether a particular protein or peptide has such activityinclude without limitation the methods described in the examplesprovided herein.

IL-13bc or active fragments thereof (IL-13bc proteins) may be fused tocarrier molecules such as immunoglobulirs. For example, soluble forms ofthe IL-13bc may be fused through “linker” sequences to the Fc portion ofan immunoglobulin. Other fusions proteins, such as those with GST, Lex-Aor MBP, may also be used.

The invention also encompasses allelic variants of the nucleotidesequences as set forth in SEQ ID NO:1 or SEQ ID NO:3, that is,naturally-ocurring alternative forms of the isolated polynucleotide ofSEQ ID NO:1 or SEQ ID NO:3 which also encode IL-13bc proteins,preferably those proteins having a biological activity of IL-13bc. Alsoincluded in the invention are isolated polynucleotides which hybridizeto the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 underhighly stringent conditions (for example, 0.1×SSC at 65° C.). Isolatedpolynucleotides which encode IL-13bc proteins but which differ from thenucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 by virtue ofthe degeneracy of the genetic code are also encompassed by the presentinvention. Variations in the nucleotide sequence as set forth in SEQ IDNO:1 or SEQ ID NO:3 which are caused by point mutations or by inducedmodifications are also included in the invention.

The present invention also provides polynucleotides encoding homologuesof the murine and human IL-13bc from other animal species, particularlyother mammalian species. Species homologues can be identified andisolated by making probes or primers from the murine or human sequencesdisclosed herein and screening a library from an appropriate species,such as for example libraries constructed from PBMCs, thymus or testisof the relevant species.

The isolated polynucleotides of the invention may be operably linked toan expression control sequence such as the pNM or pED expression vectorsdisclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), inorder to produce the IL-13bc protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185,537-566 (1990). As defined herein“operably linked” means enzymatically or chemically ligated to form acovalent bond between the isolated polynucleotide of the invention andthe expression control sequence, in such a way that the IL-13bc proteinis expressed by a host cell which has been transformed (transfected)with the ligated polynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the IL-13bc protein. Any cell type capable of expressing functionalIL-13bc protein may be used. Suitable mammalian host cells include, forexample, monkey COS cells, Chinese Hamster Ovary (CHO) cells, humankidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3cells, CV-1 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK,Rat2, BaF3, 32D, FDCP-1, PC12, M1x or C2C12 cells.

The IL-13bc protein may also be produced by operably linking theisolated polynucleotide of the invention to suitable control sequencesin one or more insect expression vectors, and employing an insectexpression system. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, e.g.,Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference. Soluble forms of the IL-13bc proteinmay also be produced in insect cells using appropriate isolatedpolynucleotides as described above.

Alternatively, the IL, 13bc protein may be produced in lower eukaryotessuch as yeast or in prokaryotes such as bacteria Suitable yeast strainsinclude Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces strains, Candida, or any yeast strain capable ofexpressing heterologous proteins. Suitable bacterial strains includeEscherichia coli, Bacillus subtilis, Salmonella typhimurium, or anybacterial strain capable of expressing heterologous proteins.

Expression in bacteria may result in formation of inclusion bodiesincorporating the recombinant protein. Thus, refolding of therecombinant protein may be required in order to produce active or moreactive material. Several methods for obtaining correctly foldedheterologous proteins from bacterial inclusion bodies are known in theart. These methods generally involve solubilizing the protein from theinclusion bodies, then denaturing the protein completely using achaotropic agent. When cysteine residues are present in the primaryamino acid sequence of the protein, it is often necessary to accomplishthe refolding in an environment which allows correct formation ofdisulfide bonds (a redox system). General methods of refolding aredisclosed in Kohno, Meth. Enzym., 185:187-195 (1990). EP 0433225 andcopending application U.S. Ser. No. 08/163,877 describe otherappropriate methods.

The IL-13bc protein of the invention may also be expressed as a productof transgenic animals, e.g., as a component of the milk of transgeniccows, goats, pigs, or sheep which are characterized by somatic or germcells containing a polynucleotide sequence encoding the IL-13bc protein.

The IL-13bc protein of the invention may be prepared by growing aculture transformed host cells under culture conditions necessary toexpress the desired protein. The resulting expressed protein may then bepurified from the culture medium or cell extracts. Soluble forms of theIL-13bc protein of the invention can be purified from conditioned media.Membrane-bound forms of IL-13bc protein of the invention can be purifiedby preparing a total membrane fraction from the expressing cell andextracting the membranes with a non-ionic detergent such as TritonX-100.

The IL-13bc protein can be purified using methods known to those skilledin the art. For example, the IL-13bc protein of the invention can beconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) or polyetheyleneimine(PEI) groups. The matrices can be acrylamide, agarose, dextran,cellulose or other types commonly employed in protein purification.Alternatively, a cation exchange step can be employed. Suitable cationexchangers include various insoluble matrices comprising sulfopropyl orcarboxymethyl groups. Sulfopropyl groups are preferred (e.g.,S-Sepharose® columns). The purification of the IL-13bc protein fromculture supernatant may also include one or more column steps over suchaffinity resins as concanavalin A-agarose, heparin-toyopearl® orCibacrom blue 3GA Sepharose®; or by hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether, or by immunoaffinity chromatography. Finally, one or morereverse-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, e.g., silica gel having pendantmethyl or other aliphatic groups, can be employed to further purify theIL-13bc protein. Affinity columns including IL-13 or fragments thereofor including antibodies to the IL-13bc protein can also be used inpurification in accordance with known methods. Some or all of theforegoing purification steps, in various combinations or with otherknown methods, can also be employed to provide a substantially purifiedisolated recombinant protein. Preferably, the isolated IL-13bc proteinis purified so that it is substantially free of other mammalianproteins.

IL-13bc proteins of the invention may also be used to screen for agentswhich are capable of binding to IL-13bc or IL-13R or which interferewith the binding of IL-13 to the IL-13 or IL-13bc (either theextracellular or intracellular domains) and thus may act as inhibitorsof normal binding and cytokine action (“IL-13R inhibitors”). Bindingassays using a desired binding protein, immobilized or not, are wellknown in the art and may be used for this purpose using the IL-13bcprotein of the invention. Purified cell based or protein based (cellfree) screening assays may be used to identify such agents. For example,IL-13bc protein may be immobilized in purified form on a carrier andbinding to purified IL-13bc protein may be measured in the presence andin the absence of potential inhibiting agents. A suitable binding assaymay alternatively employ a soluble form of IL-13bc of the invention.Another example of a system in which inhibitors may be screened isdescribed in Example 2 below.

In such a screening assay, a first binding mixture is formed bycombining IL-13 or a fragment thereof and IL-13bc protein, and theamount of binding in the first binding mixture (B_(o)) is measured. Asecond binding mixture is also formed by combining IL-13 or a fragmentthereof, IL-13bc protein, and the compound or agent to be screened, andthe amount of binding in the second binding mixture (B) is measured. Theamounts of binding in the first and second binding mixtures arecompared, for example, by performing a calculation of the ratio B/B_(o).A compound or agent is considered to be capable of inhibiting binding ifa decrease in binding in the second binding mixture as compared to thefirst binding mixture is observed. Optionally, the second chain ofIL-13R can be added to one or both of the binding mixtures. Theformulation and optimization of binding mixtures is within the level ofskill in the art, such binding mixtures may also contain buffers andsalts necessary to enhance or to optimize binding, and additionalcontrol assays may be included in the screening assay of the invention.

Compounds found to reduce the binding activity of IL-13bc protein toIL-13 or its fragment to any degree, preferably by at least about 10%,more preferably greater than about 50% or more, may thus be identifiedand then secondarily screened in other binding assays and in vivoassays. By these means compounds having inhibitory activity for LI-13bcbinding which may be suitable as therapeutic agents may be identified.

IL-13bc proteins, and polynucleotides encoding them, may also be used asdiagnostic agents for detecting the expression or presence of IL-13bc,IL-13R, IL-13 or cells expressing IL-13bc, IL-13R or IL-13. The proteinsor polynucleotides may be employed for such purpose in standardprocedures for diagnostics assays using these types of materials.Suitable methods are well known to those skilled in the art.

As used herein “IL-13R” refers to IL-13bc and/or a second IL-13 receptorchain known as “IL-3Rα1” or “NR4” (see: murine receptor chain, Hilton etal., Proc. Natl. Acad. Sci. USA 1996, 93:497-501; human receptor chain,Aman et al., J. Biol. Chem. 1996, 271:29265-70, and Gauchat et al., Eur.J. Immunol. 1997, 27:971-8).

IL-13bc acts as a mediator of the known biological activities of IL-13.As a result, IL-13bc protein (particularly, soluble IL-13bc proteins),IL-13R inhibitors (i.e., antagonists of interaction of IL-13 with IL-13R(such as, for example, antibodies to IL-13R (including particularly toIL-13bc or to IL-13Rα1) and fragments thereof, antibodies to IL-13 andfragments thereof, soluble IL-13Rα1 proteins, and small molecule andother inhibitors of the interaction of IL-13 with IL-13R (including withIL-13bc and/or with IL-13Rα1) may be useful in treatment or modulationof various medical conditions in which IL-13 is implicated or which areeffected by the activity (or lack thereof) of IL-13 (collectively“IL-13-related conditions”). Mutated forms of IL-4 which bind to IL-13Rcan also be used as IL-13 antagonists (see, for example, those disclosedin Shanafelt et al., Proc. Natl. Acad. Sci. USA 1998, 95:9454-8; Aversaet al., J. Exp. Med. 1993, 178:2213-8; and Grunewald et al., J. Immunol.1998, 160:4004-9).

IL-13-related conditions include without limitation Ig-mediatedconditions and diseases, particularly IgE-mediated conditions (includingwithout limitation atopy, allergic conditions, asthma, immune complexdiseases (such as, for example, lupus, nephrotic syndrome, nephritis,glomerulonephritis, thyroiditis and Grave's disease)); inflammatoryconditions of the lungs; immune deficiencies, specifically deficienciesin hematopoietic progenitor cells, or disorders relating thereto; cancerand other disease. Such pathological states may result from disease,exposure to radiation or drugs, and include, for example, leukopenia,bacterial and viral infections, anemia, B cell or T cell deficienciessuch as immune cell or hematopoietic cell deficiency following a bonemarrow transplantation. Since IL-13 inhibits macrophage activation,IL-13bc proteins may also be useful to enhance macrophage activation(i.e., in vaccination, treatment of mycobacterial or intracellularorganisms, or parasitic infections).

IL-13bc proteins may also be used to potentiate the effects of IL-13 invitro and in vivo. For example, an IL-13bc protein can be combined witha protein having IL-13 activity (preferably IL-13) and the resultingcombination can be contacted with a cell expressing at least one chainof IL-13R other than IL-13bc (preferably all chains of IL-13R other thanIL-13bc, such as IL-13Rα1). Preferably, the contacting step is performedby administering a therapeutically effective amount of such combinationto a mammalian subject in vivo. The pre-established association of theIL-13 protein with the IL-13bc protein will aid in formation of thecomplete IL-13/IL-13R complex necessary for proper signaling. See forexample the methods described by Economides et al., Science 270:1351(1995).

IL-13bc protein and IL-13R inhibitors, purified from cells orrecombinantly produced, may be used as a pharmaceutical composition whencombined with a pharmaceutically acceptable carrier. Such a compositionmay contain, in addition to IL-13bc or inhibitor and carrier, variousdiluents, fillers, salts, buffers, stabilizers, solubilizers, and othermaterials well known in the art. The term “pharmaceutically acceptable”means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration.

The pharmaceutical composition of the invention may also containcytokines, lymphokines, or other hematopoietic factors such as M-CSF,GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-14, IL-15, G-CSF, stem cell factor, and erythropoietin.The pharmaceutical composition may also include anti-cytokineantibodies. The pharmaceutical composition may contain thrombolytic oranti-thrombotic factors such as plasminogen activator and Factor VIII.The pharmaceutical composition may further contain otheranti-inflammatory agents. Such additional factors and/or agents may beincluded in the pharmaceutical composition to produce a synergisticeffect with isolated IL-13bc protein or IL-13bc inhibitor, or tominimize side effects caused by the isolated IL-13bc or IL-13bcinhibitor. Conversely, isolated IL-13bc or IL-13bc inhibitor may beincluded in formulations of the particular cytokine, lymphokine, otherhematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent to minimize side effects of the cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent.

The pharmaceutical composition of the invention may be in the form of aliposome in which isolated IL-13bc protein or IL-13bc inhibitor iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layerswhich in aqueous solution. Suitable lipids for liposomal formulationinclude, without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S.Pat. No. 4,501,728: U.S. Pat. No. 4,837,028; and U.S. Pat. No.4,737,323, all of which are incorporated herein by reference.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, e.g.,amelioration of symptoms of, healing of, or increase in rate of healingof such conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of isolated IL-13bc protein or IL-13bcinhibitor is administered to a mammal. Isolated IL-13bc protein orIL-13bc inhibitor may be administered in accordance with the method ofthe invention either alone or in combination with other therapies suchas treatments employing cytokines, lymphokines or other hematopoieticfactors. When co-administered with one or more cytokines, lymphokines orother hematopoietic factors, IL-13bc protein or IL-13bc inhibitor may beadministered either simultaneously with the cytokine(s), lymphokine(s),other hematopoietic factor(s), thrombolytic or anti-thrombotic factors,or sequentially. If administered sequentially, the attending physicianwill decide on the appropriate sequence of administering IL-13bc proteinor IL-13bc inhibitor in combination with cytokine(s), lymphokine(s),other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

Administration of IL-13bc protein or IL-13bc inhibitor used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, or cutaneous, subcutaneous, or intravenousinjection. Intravenous administration to the patient is preferred.

When a therapeutically effective amount of IL-13bc protein or IL-13bcinhibitor is administered orally, IL-13bc protein or IL-13bc inhibitorwill be in the form of a tablet, capsule, powder, solution or elixir.When administered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%IL-13bc protein or IL-13bc inhibitor, and preferably from about 25 to90% IL-13bc protein or IL-13bc inhibitor. When administered in liquidform, a liquid carrier such as water, petroleum, oils of animal or plantorigin such as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of IL, 13bc protein or IL-13bc inhibitor, and preferably fromabout 1 to 50% IL-13bc protein or IL-13bc inhibitor.

When a therapeutically effective amount of IL-13bc protein or IL-13bcinhibitor is administered by intravenous, cutaneous or subcutaneousinjection, IL-13bc protein or IL-13bc inhibitor will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to IL-13bc proteinor IL-13bc inhibitor an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additive known to those of skill in the art.

The amount of IL-13bc protein or IL-13bc inhibitor in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of IL-13bc protein or IL-13bc inhibitorwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of IL-13bc protein or IL-13bcinhibitor and observe the patient's response. Larger doses of IL-13bcprotein or IL-13bc inhibitor may be administered until the optimaltherapeutic effect is obtained for the patient, and at that point thedosage is not generally increased further. It is contemplated that thevarious pharmaceutical compositions used to practice the method of thepresent invention should contain about 0.1 μg to about 100 mg of IL-13bcprotein or IL-13bc inhibitor per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the IL-13bc protein or IL-13bc inhibitorwill be in the range of 12 to 24 hours of continuous intravenousadministration. Ultimately the attending physician will decide on theappropriate duration of intravenous therapy using the pharmaceuticalcomposition of the present invention.

IL-13bc proteins of the invention may also be used to immunize animalsto obtain polyclonal and monoclonal antibodies which specifically reactwith the IL-13bc protein and which may inhibit binding of IL-13 orfragments thereof to the receptor. Such antibodies may be obtained usingthe entire IL-13bc as an immunogen, or by using fragments of IL-13bc,such as the soluble mature IL-13bc. Smaller fragments of the IL-13bc mayalso be used to immunize animals. The peptide immunogens additionallymay contain a cysteine residue at the carboxyl terminus, and areconjugated to a hapten such as keyhole limpet hemocyanin (KLH).Additional peptide immunogens may be generated by replacing tyrosineresidues with sulfated tyrosine residues. Methods for synthesizing suchpeptides are known in the art, for example, as in R. P. Merrifield,J.Amer.Chem.Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBSLett. 211, 10 (1987).

Neutralizing or non-neutralizing antibodies (preferably monoclonalantibodies) binding to IL-3bc protein may also be useful therapeuticsfor certain tumors and also in the treatment of conditions describedabove. These neutralizing monoclonal antibodies may be capable ofblocking IL-13 binding to the IL-13bc.

EXAMPLE 1 Isolation of IL-13bc cDNAs

Isolation of the Murine IL-13 Receptor Chain.

5 ug of polyA+ RNA was prepared from the thymuses of 6-8 week oldC3H/HeJ mice. Double stranded, hemimethylated cDNA was prepared usingStratagene's cDNA synthesis kit according to manufacturers instructions.Briefly, the first strand was primed with an oligodT-Xho primer, andafter second strand synthesis, EcoRI adapters were added, and the cDNAwas digested with XhoI and purified. The cDNA was ligated to theXhoI-EcoRI sites of the Zap Express (Stratagene) lambda vector, andpackaged using Gigapak II Gold packaging extracts (Stratagene) accordingto the manufacturers instructions. A library of 1.5×10⁶ resultingrecombinant phage was amplified following manufacturer's instructions.This library was screened with a degenerate 17mer oligonucleotide probeof the sequence KSRCTCCABK CRCTCCA (SEQ ID NO:5) (K=G+T; S=C+G; R=A+G;B=C+G+T) using standard TMAC hybridization conditions as described(Current Protocols in Molecular Biology, Ausubel, et al., editors., JohnWiley and Sons, 1995, section 6.4.3). Clone A25 was identified becauseit hybridized to the 17mer probe, but not to probes derived from knownhematopoietin receptors. This clone was isolated in plasmid form fromthe ZapExpress vector as per manufacturers instruction, and the DNAsequence was determined. The DNA sequence encoded a novel member of thehematopoietin receptor family.

Clone A25 containing the polynucleotide having the sequence of SEQ IDNO:1 was deposited with ATCC as pA25pBKCMV at accession number 69997 onFeb. 22, 1996.

Isolation of the Human IL-13 Receptor Chain.

A partial fragment of the human homolog of the murine receptor wasisolated by PCR using oligonucleotides derived from the murine sequence.cDNA was prepared from human testis polyA+ RNA that was obtained fromClontech. A DNA fragment of 274 base pairs was amplified from this cDNAby PCR with the following oligonucleotides: ATAGTFAAACCATTGCCACC (SEQ IDNO:6) and CTCCATrCGCTCCAAATTCC (SEQ ID NO:7) using AmpliTaq polymerase(Promega) in 1× Taq buffer containing 1.5 mM MgCl2 for 30 cycles ofincubation (94° C.×1 minute, 42° C. for 1 minute, and 72° C. for 1minute). The DNA sequence of this fragment was determined, and twooligonucleotides were prepared from an internal portion of this fragmentwith the following sequence: AGTCTATCTTACTTTTACTCG (SEQ ID NO:8) andCATCTGAGCAATAAATATTCAC (SEQ ID NO:9). These oligonucleotides were usedas probes to screen a human testis cDNA library purchased from CLONTECH(cat #HL1161). Filters were hybridized at 52° C. using standard 5×SSChybridization conditions and washed in 2×SSC at 52° C. Twenty two cloneswere isolated that hybridized to both oligonucleotides in a screen of400,000 clones. DNA sequence was determined from four of the cDNAclones, and all encoded the same novel hematopoietin receptor. Thepredicted DNA sequence of the fall length human receptor chain is shownas SEQ ID NO:3.

The human clone was deposited with ATCC as phA25#11pDR2 at accessionnumber 69998 on Feb. 22, 1996.

EXAMPLE 2 Expression of Soluble IL-13bc Protein and Assay of Activity

Production and Purification of Soluble IL-13bc-Ig.

DNA encoding amino acids 1-331 of the extracellular domain of murineIL-13bc was fused to a spacer sequence encoding gly-ser-gly by PCR andligated in frame with sequences encoding the hinge CH2 CH3 regions ofhuman IgG1 of the COS-1 expression vector pED.Fc. IL-13bc-Ig wasproduced from DEAE-dextran transfected COS-1 cells and purified viaprotein A sepharose chromatography (Pharmacia).

B9 Proliferation Assay

Stimulation of proliferation of B9 cells (Aarden et al. Eur. J. Immunol.1987. 17:1411-1416) in response to IL-13 or IL-4 was measured by3H-thymidine incorporation into DNA. Cells (5×103/well) were seeded into96 well plates with media containing growth factors at varyingconcentrations in the presence or absence of II13bc-Ig at lug/ml. Afterincubation for 3 days 1 uCi/well of 3H-thymidine was added and the cellsincubated for an additional 4 hrs. Incorporated radioactivity wasdetermined using a LKB 1205 Plate reader.

The B9 cell line proliferated in response to IL-13, IL-4 or IL6. Onlyresponses to IL-13 were inhibited by the soluble IL-13bc-Ig, indicatingthat this receptor binds IL-13 specifically, but not IL-4 or IL-6. Thetables show cpm. Two separate experiments are shown. IL-13 plus IL-4plus cytokine IL-13 A25-Fc IL-4 A25-Fc Cos IL-6 dilution (3 ng/ml) (1ug/ml) (20 ng/ml) (1 ug/ml) (1/10,000) 1 37734 1943 6443 6945 37887 1/330398 1571 2680 2442 36500 1/10 16101 1461 1767 1771 33335 1/30 21481567 1619 1783 27271 1/100 1574 1419 1522 1576 18831 1/300 1512 15311373 1577 7768 1/1000 1316 1392 1190 1474 2760 1/3000 1834 1994 14821819 1672

IL-13 plus IL-4 plus Cos IL-6 cytokine IL-13 A25-Fc IL-4 A25-Fc Cos IL-6plus A25-Fc dilution (3 ng/ml) (5 ug/ml) (20 ng/ml) (5 ug/ml) (1/10,000)(5 ug/ml) 1 6413 295 1216 1158 6969 7703 1/3 5432 281 518 656 7827 88041/10 2051 281 489 520 8345 10027 1/30 506 319 279 476 8680 9114 1/100430 372 288 423 7426 10364 1/300 330 287 323 420 5531 6254 1/1000 326389 348 nt 2524 nt no cytokine 339 279 404 394 326 279

EXAMPLE 3 Direct Binding of Soluble IL-13bc to IL-13 Measured by SurfacePlasmon Resonance (Biacore Analysis)

A Biacore biosensor was used to measure directly the specific binding ofIL-13 to purified IL-13bc-Ig (Pharmacia, Johnsson et al., 1991).Approximately 10,000 to 17,000 resonance units (RU) of purifiedIL-13bc-Ig, human IgG1 or irrelevant receptor were each covalentlyimmobilized to different flow cells on the sensor chip as recommended bythe manufacturer. (RU's are a refelction of the mass of protein bound tothe sensor chip surface.) Purified IL-13 was injected across the flowcells at 5 ul/min for 10 rains in the presence or absence of excesspurified IL-13bc-Ig. Binding was quantified as the difference in RUbefore and after sample injection. Specific II 13 binding of 481.9 RUwas observed only for immobilized IL-13bc-Ig whereas coinjection ofIL-13 plus IL-13bc-Ig resulted in no binding to the immobilizedIL-13bc-Ig (4 RU). No IL-13 binding was observed for either immobilizedIgG or IL-11R-Ig (5.4 and 3.7 RU respectively). IL-13bc-Ig IgG controlIL-11R-Ig Sample (10,383 RU) (13,399 RU) (17,182 RU) 100 ng/ml human481.9 RU bound 5.4 RU bound 3.7 RU bound IL-13 100 ng/ml human 4.0 RUbound not tested not tested IL-13 + soluble IL-13bc-Ig

EXAMPLE 4 Binding of IL-13 Expressed in COS Cells to Labeled IL-13BC-IgFusion Protein: COS in situ Detection of IL-13 with IL-13bc-Fc

Expression vectors for IL-13, IL 4, IL-11 or empty vector weretransfected into COS-1 cells in duplicated plates via the DEAE-dextranmethod. Two days after transfection cells were washed twice in phosphatebuffered saline (PBS) and fixed in the culture dish for 10′ at 4° C.with methanol. Following fixation cells were washed twice with PBS thenrinsed once with binding buffer (PBS, 1% (w/v) bovine serum albumin,).1%(w/v) sodium azide) and incubated for two hours at 4° C. in bindingbuffer with IL-13bc-Fc at 1.0 ug/rnl or with relevant anti-cytokineantisera. Cells were washed twice with PBS and incubated at 4° C. withshaking in alkaline phosphatase labeled Rabbit F(ab)2′ anti-human IgGdiluted 1:500 in binding buffer (for Fc fusion detection) or RabbitF(ab)2′ anti-rat IgG (for anti-cytokine detection). Cells were againwashed twice in PBS. Alkaline phosphatase activity was visualized usingnitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate.

Specific binding was visualized under the microscope. Only cellstransfected with IL-13 showed specific binding to IL-13bc-Ig. (see photoof transfected cells, the Figure).

EXAMPLE 5 Other Systems for Determination Biological Activity of IL-13bcProtein

Other systems can be used to determine whether a specific IL-13bcprotein exhibits a “biological activity” of IL-13bc as defined herein.The following are examples of such systems.

Assays for IL-13 Binding

The ability of a IL-13bc protein to bind IL-13 or a fragment thereof canbe determine by any suitable assays which can detect such binding. Somesuitable examples follow.

Binding of IL-13 to the extracellular region of the IL-13bc protein willspecifically cause a rapid induction of phosphotyrosine on the receptorprotein. Assays for ligand binding activity as measured by induction ofphosphorylation are described below.

Alternatively, a IL-13bc protein (such as, for example, a soluble formof the extracellular domain) is produced and used to detect IL-13binding. For example, a DNA construct is prepared in which theextracellular domain (truncated prior, preferably immediately prior, tothe predicted transmembrane domain) is ligated in frame to a cDNAencoding the hinge C_(H)2 and C_(H)3 domains of a human immunoglobulin(Ig) γ1. This construct is generated in an appropriate expression vectorfor COS cells, such as pEDΔC or pMT2. The plasmid is transientlytransfected into COS cells. The secreted IL-13bc-Ig fusion protein iscollected in the conditioned medium and purified by protein Achromatography.

The purified IL-13bc-Ig fusion protein is used to demonstrate IL-13binding in a number of applications. IL-13 can be coated onto thesurface of an enzyme-linked immunosorbent assay (ELISA) plate, and thenadditional binding sites blocked with bovine serum albumin or caseinusing standard ELISA buffers. The IL-13bc-Ig fusion protein is thenbound to the solid-phase IL-13, and binding is detected with a secondarygoat anti-human Ig conjugated to horseradish peroxidase. The activity ofspecifically bound enzyme can be measured with a colorimetric substrate,such as tetramethyl benzidine and absorbance readings.

IL-13 may also be expressed on the surface of cells, for example byproviding a transmembrane domain or glucosyl phosphatidyl inositol (GPI)linkage. Cells expressing the membrane bound IL-13 can be identifiedusing the IL-13bc-Ig fusion protein. The soluble IL-13bc-Ig fusion isbound to the surface of these cells and detected with goat anti-human Igconjugated to a fluorochrome, such as fluorescein isothiocyanate andflow cytometry.

Interaction Trap

A yeast genetic selection method, the “interaction trap” [Gyuris et al,Cell 75:791-803, 1993], can be used to determine whether a IL-13bcprotein has a biological activity of IL-13bc as defined herein. In thissystem, the expression of reporter genes from both LexAop-Leu2 andLexAop-LacZ relies on the interaction between the bait protein, forexample in this case a species which interacts with human IL-13bc, andthe prey, for example in this case the human IL-13bc protein. Thus, onecan measure the strength of the interaction by the level of Leu2 or LacZexpression. The most simple method is to measure the activity of theLacZ encoded protein, β-galactosidase. This activity can be judged bythe degree of blueness on the X-Gal containing medium or filter. For thequantitative measurement of β-galactosidase activity, standard assays&an be found in “Methods in Yeast Genetics” Cold Spring Harbor, N.Y.,1990 (by Rose, Md., Winston, F., and Hieter, P.).

In such methods, if one wishes to determine whether the IL-13bc proteininteracts with a particular species (such as, for example, a cytosolicprotein which binds to the intracellular domain of the IL-13bc in vivo),that species can be used as the “bait” in the interaction trap with theIL-13bc protein to be tested serving as the “prey”, or vice versa.

EXAMPLE 6 Treatment of Asthma Using Soluble IL-13bc Protein

A well-characterized murine model of allergic asthma was used, in whichallergen exposure leads to airway hyper responsiveness (“AFHR”),pulmonary eosinophilia, elevations in antigen-specific serum IgE levels,and increases in airway epithelial mucus content (3, 11). Male A/J micewere immunized intraperitoneally and subsequently challengedintratracheally with soluble ovalbumin (OVA), the allergic phenotypebeing assessed 4 days after antigen challenge (13). Blockade of IL-13was performed by the systemic administration of a soluble IL-13bc-IgGFcfusion protein (sIL-13bc-Fc), which specifically binds to andneutralizes IL-13, 24 hours before subsequent intratracheal allergenchallenge (14). Challenge of allergen-immunized mice resulted insignificant increases in airway responsiveness to acetylcholine (15)(FIG. 2A). Blockade of IL-13 resulted in complete reversal of suchestablished allergen-induced AHR; thus IL-13 is necessary for theexpression of AHR in this model. The ability of IL-13 ablation toreverse AHR after full development of the phenotype of allergic asthmacontrasts with the inability of IL-4 ablation to accomplish such areversal. The mechanism underlying the effectiveness of IL-4Rα blockadein reversing allergen-induced AHR may be the inhibition ofIL-13-mediated processes, consistent with the fact that Stat6 activationis downstream of IL4Rα-mediated signaling for both cytokines. IL-13 isprobably the primary CD4+ T cell-derived factor responsible forallergen-induced AHR.

To evaluate candidate mechanisms underlying IL-13-dependent expressionof AHR, we characterized known allergic effector cascades. Eosinophilshave been implicated as primary effector cells in asthma and asthmaticAHR (16), but inhibition of IL-13 prior to repeat antigen provocationdid not significantly affect allergen-induced pulmonary eosinophilia(17) (FIG. 2B). To assess the relevance of IgE-mediated pathways, wemeasured OVA-specific serum IgE (18). OVA-specific levels of IgE wereobserved in OVA-sensitized and -challenged mice, whereas noantigen-specific antibody levels were detected in PBS-immunized and-challenged mice (FIG. 2C). Blockade of IL-13 did not alter OVA-specificIgE levels, a lack of suppression which is likely due to the fact thatIL-13 blockade occurred after initial antigen priming and antibodyformation. Nonetheless, these results show that AHR is not dependentupon IgE production in this model, consistent with reports that allergicAHR develops normally in IgE deficient and B cell deficient mice (19).

In congruence with the pathology of human asthma, allergic asthma inmurine models is associated with a marked increase in the mucus contentof the airway epithelium (5, 11). Mucus hypersecretion is particularlyprofound in autopsy specimens from patients who die of acute asthmaattacks (20). Blockade of IL-13 reverses allergen-induced increases inmucus-containing cells in the airways (FIG. 3), demonstrating thatallergen-induced increases in airway mucus content are dependent uponIL-13. IL-4 is also implicated in this process, as IL-4 transgenic micedisplay marked goblet cell hyperplasia in the absence of antigensensitization (5). However, transfer of Th2 clones from bothIL-4-deficient and control mice into murine airways induces mucusoverproduction (21), suggesting, yet again, that the immunoregulatoryrole of IL-4 needs to be carefully differentiated from its role as aneffector molecule.

Daily administration of recombinant IL-13 (rIL-13) to the airways ofnaive (unimmunized) mice induced AFR, demonstrating that increases inIL-13 activity were sufficient to induce AHR (FIG. 4A) (22). AHRdeveloped by 72 hours after the start of rIL-13 administration. Asignificant influx of eosinophils into bronchoalveolar lavage fluid wasobserved early after rIL-13 administration, however pulmonaryeosinophilia was not observed at the time of expression of AHR (FIG.4B). Although the significance of the time course of eosinophil influxremains unclear, it suggests that IL-13 alone may be sufficient toinitiate eosinophilic infiltration of the airways, perhaps through itsability to upregulate chemokine expression (23). Airway administrationof rIL-13 also resulted in a time-dependent increase in total serum IgE(FIG. 4C) (24), in line with the previously-reported ability of IL-13 toregulate IgE synthesis (25). Increases in serum IgE were independent ofany immunization with allergen, findings that resonate with theobservation that the human asthmatic phenotype correlates better withtotal, rather than allergen-specific, serum IgE concentrations (26). Aspredicted from the above IL-13 inhibition studies, the administration ofrIL-13 induced an increase in airway mucus production (FIG. 4D) (27).

REFERENCES AND NOTES

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Mice were immunized by an    intraperitoneal injection of 10 ug ovalbumin (OVA; Crude grade IV,    Sigma; St. Louis, Mo.) in 0.2 ml PBS or PBS alone. 14 days after    immunization, mice were anesthetized with a mixture of ketamine and    xylazine (45 and 8 mg/kg, respectively) and challenged    intratracheally with 50 ul of a 1.5% solution of OVA or an    equivalent volume of PBS as a control. 10 days after this first    antigen challenge, mice were challenged again intratracheally with    either OVA or PBS. Characterization of the allergic phenotype was    performed 96 hours after the second antigen challenge.-   14. Human EL-13bc was cloned as described above. For soluble    expression of the murine homolog, a pED expression vector containing    DNA encoding the murine sIL-13bc extracellular domain, fused in    frame with the hinge CH2/CH3 regions of human IgG1 (as described in    previous examples), was transfected into CHO cells [D. D. Donaldson    et al., J. Immunol. 161, 2317 (1998)]. The sIL-13bc-Fc was purified    with rProtein A-Sepharose [J. F. Urban et al., Immunity 8, 255    (1998)]. The in vitro ID₅₀, as determined by the ability to    neutralize 3 ng/ml of murine IL-13 in the B9 proliferation assay was    approximately 10 ng/ml. Human IgG, used as a control for    sIL-13bc-Fc, was similarly purified by rProtein A-Sepharose    chromatography from a 10% solution of human immune globulin that is    commercially available for intravenous administration (Miles)    [ibid]. Mice were given sIL-13bc-Fc (400 ug), or an equivalent    amount of the control hu-IgG, by intraperitoneal injection on Day    −1, O, +1, and +3 of secondary antigen challenge.-   15. Airway reactivity to intravenous administration of acetylcholine    was measured (11), 3 days after final intratracheal challenge. Mice    were anesthetized with sodium pentobarbital (90 mg/kg), intubated,    ventilated at a rate of 120 breaths/minute with a constant tidal    volume of air (0.2 ml), and paralyzed with decamethonium bromide (25    mg/kg). After establishment of a stable airway pressure,    acetylcholine was injected intravenously (50 ug/kg) and dynamic    airway pressure was followed for 5 minutes.-   16. G. J. Gleich, J. All. Clin. Immunol. 8,422 (1990).-   17. Bronchoalveolar lavage was conducted as described (11).-   18. A kidney was excised, and pooled blood was collected for    antibody analysis as described (11). Serum was separated by    centrifugation and stored at −80° C. until analysis. Serum    OVA-specific IgE levels were determined by sandwich ELISA. Sample    wells were coated with a 0.01% OVA solution in PBS, blocked with 10%    FBS in PBS, and washed with 0.05% Tween-20 in PBS. Serum samples    were diluted 1:10 and 1:100 with 10% FBS in PBS. After an overmight    incubation, plates were washed with 0.05% Tween-20 in PBS and    biotin-conjugated anti-mouse IgE (PharMingen, San Diego, Calif.) was    added. After a wash, 0.0025 mg/ml avidin peroxidase (Sigma) in 10%    FBS/PBS was added, and plates were developed with ABTS    (2,2′-azino-did[3-ethyl-benzthiazone sulfonate]) (Kirkegaard and    Perry). Plates were read at 405 nm within 30 minutes. Reported O.D.    values are of serum samples diluted 1:10 since these values were    proven to be below the saturation point of the assay by comparison    of O.D. values of serum samples diluted 1:100 with 10% FBS/PBS.-   19. P. D. Mehihop et al., Proc. Natl. Acad. Sci. USA 94, 1344    (1997); M. Korsgren et al., J. Exp. Med. 185, 885 (1997).-   20. T. Aikawa et al., Chest 101, 916 (1992).-   21. L. Cohn, R. J. Homer, A. Marinov, J. Rankin, K. Bottomly. J.    Exp. Med. 186, 1737 (1997).-   22. DNA encoding a honeybee melittin leader [D. C. Tessier, D. Y.    Thomas, H. E. Khouri, F. Laliberte, T. Vernet, Gene 2, 177 (1991)]    followed by a six-histidine tag was fused by an enterokinase    cleavage site to the mature region of murine IL-13 at Gly21 and    constructed in the mammalian expression vector pHTop. H6-EK murine    IL-13 protein was produced from stably-transfected CHO cells and    purified via Ni-NTA chromatography to greater than 97% purity as    determined by SDS-PAGE. Protein concentration was determined by    absorption at 280 nm and endotoxin contamination was less than 30    EU/mg as measured by Cape Cod Associates LAL assay. The ED₅₀ of H6EK    murine IL-13 as determined by the Ba/F3.IL-13R 1 proliferation assay    was 1 ng/ml. Murine rIL-13 (5 ug in a total volume of 50 ul) was    administered daily by intratracheal instillation to naive mice    anesthesized with a mixture of ketamine and xylazine (45 and 8    mg/kg, respectively).-   23. M. Goebeler et al., Immunol. 91, 450 (1997).-   24. A murine IgE-specific ELISA was used to quantitate total IgE    immunoglobulin levels in serum using complementary antibody pairs    for mouse IgE (R35-72 and R35-92) obtained from PharMingen according    to the manufacturer's instructions. Duplicate samples (of a 1/10    dilution in 10% FBS in PBS) were examined from each animal. O.D.    readings of samples were converted to pg/ml using values obtained    from standard curves generated with known concentrations of    recombinant mouse IgE (5-2000 pg/ml), and the final concentration    was obtained by multiplying by the dilution factor.-   25. C. L. Emson, S. E. Bell, A. Jones, W. Wisden, A. N.J.    McKenzie, J. Exp. Med. 188, 399 (1998).-   26. L. R. Friedhoff, D. G. Marsh, Int. Arch. All. Immunol. 100, 355    (1993).-   27. To examine the effects of rIL-13 on mucus cell content of the    airway epithelium, lungs were excised and fixed in 10% formalin.    They were then washed in 70% ethanol, dehydrated, embedded in glycol    methacrylate, cut into 10 UM sections, mounted on slides, and    stained with hematoxylin and eosin and periodic acid Schiff. Four    sections were examined per animal; 4 fields were scored per lung    section. Sections were scored on a scale from 1-4 with 1    representing no mucus cell content.-   28. J. Luyimbazi, X. Xu, M. Wills-Karp, unpublished results.-   29. C. Walker et al., Am. Rev. Respir. Dis. 146, 109 (1992); M.    Humbert et al., J. All. Clin. Immunol. 99, 657 (1997); S. K.    Huang, J. Immunol. 155, 2688 (1995).-   30. D. G. Marsh, et al., Science 264, 1152 (1996).-   31. L. J. Rosenwasser. N. Engl. J. Med. 337, 1766 (1977).-   32. G. K. Hershey et al., N Engl. J. Med. 337, 1720 (1997).

All patent and literature references cited herein are incorporated byreference as if fully set forth.

1-47. (canceled)
 48. A method of ameliorating asthma in a mammaliansubject, said method comprising administering to said subject aneffective amount of an antibody to IL-13 or an IL-13 binding fragmentthereof, thereby treating asthma in said subject.
 49. The method ofclaim 48, wherein said method comprises administering a pharmaceuticalcomposition comprising an antibody to IL-13.
 50. The method of claim 48,wherein said method comprises administering a pharmaceutical compositioncomprising an IL-13 binding fragment of an antibody to IL-13.
 51. Themethod of claim 48, The wherein said antibody or IL-13 binding fragmentthereof antagonizes binding of IL-13 to a human IL-13bc.
 52. The methodof claim 48, wherein said composition is administered by intravenous,cutaneous, subcutaneous, or injection.
 53. The method of claim 48,wherein said composition is administered by intravenous injection. 54.The method of claim 48, wherein said antibody is administered for about12 to 24 hours of continuous administration.
 55. The method of claim 48,wherein said composition is administered at a dose of about 0.1 μg toabout 100 mg per kg body weight.
 56. A method of amelioratingallergen-induced airway hyper responsiveness in a mammalian subject,said method comprising administering to said subject an effective amountof an antibody to IL-13 or an IL-13 binding fragment thereof, therebytreating allergen-induced airway hyper responsiveness in said subject.57. The method of claim 56, wherein said method comprises administeringa pharmaceutical composition comprising an antibody to IL-13.
 58. Themethod of claim 56, wherein said method comprises administering apharmaceutical composition comprising an IL-13 binding fragment of anantibody to IL-13.
 59. The method of claim 56, wherein said IL-13antibody or IL-13 binding fragment thereof antagonizes binding of IL-13to a human IL-13bc.
 60. The method of claim 56, wherein said compositionis administered by intravenous, cutaneous, or subcutaneous injection.61. The method of claim 56, wherein said antibody is administered forabout 12 to 24 hours of continuous administration.
 62. The method ofclaim 56, wherein said composition is administered at a dose of about0.1 μg to about 100 mg per kg body weight.